The high mortality for lung cancer urgently requires the availability of new, noninvasive diagnostic tools for use in screening and prevention programs. Our long term goal is to develop screening approach for lung cancer. Silencing of tumor suppressor genes by aberrant promoter hypermethylation is recognized as a crucial component in lung cancer initiation and progression. Recently, we have been able to map gene promoter methylation in DNA samples recovered from EBC. However, the number of genes that can be analyzed in any given EBC sample is limited by the quantity of DNA template. Therefore, improvement in this method to multiplex the assay is necessary before expanding validation testing. The objective in this R03 proposal is to improve the method for multiple gene methylation analysis in exhaled breath condensate. We hypothesize that the current method can be modified by the addition of a Whole Genome Amplification (WGA) step to make it possible to perform multiple gene methylation analyses in EBC samples. To validate the utility of the developed method, we will analyze the methylation status, with and without WGA, of p16, DAPK, RASSF1A, MGMT, PAX51, PAX52 and CDH1 in sixty EBC samples from smokers and non-smokers, and also compare EBC with sputum from the same subjects, for reference. Our rationale for these studies is that their successful completion would provide the research community with a new, non-invasive method for further clinical studies that evaluate DNA methylation as a biomarker for risk assessment, or early detection, of lung cancer. Project Narrative: The high mortality from lung cancer warrants the development of new, noninvasive diagnostic tools for use in screening and prevention programs. Recently, we have been able to assess gene promoter methylation, a cancer biomarker, in DNA samples recovered from exhaled breath. The objective in this R03 proposal is to improve the method for multiple gene methylation analysis in exhaled breath condensate, to provide the research community with a new, non-invasive method for assessing an individual's risk for lung cancer.